CCL2/CCR2 dependence of perilymphatic cellular accumulation in D6-deficient mice. (A) Confocal images representing the localization of CCL2 (red) on Lyve1⁺ lymphatic vessels (green) surrounding the subcapsular sinus of a popliteal LN of a D6-deficient mouse 24 hours after subcutaneously footpad injection of LPS. DAPI (blue) was used for nuclear staining (blue). LECs showing Lyve1 and CCR2 positivity are marked by yellow arrows. Images were acquired at ×63 magnification with a scan zoom at 2.0 on a Plan-Apochromat 63×/1.4 oil Ph3 objective. Scale bar represents 20 μm. (B) CD11b⁺Gr1^hi cells express CCR2. Popliteal LN cells collected from D6-deficient mice, 24 hours after footpad LPS injection, were incubated with AF-647–labeled human (h) CCL2, in the presence or absence of a 10-fold molar excess of unlabeled hCCL2 at 37°C for 30 minutes, and analyzed by flow cytometry. The data show CCL2 binding by CD11b⁺Gr1^hi but less by CD11b⁺Gr1^low cells. Cells that were left unstained were used as a control for background fluorescence. (C) CCR2 is involved in the accumulation of myelomonocytic cells in D6-deficient LNs. Two representative contour plots showing the ability of a CCR2 blocker (RS504393) to reduce numbers of CD11b⁺Gr1⁺ cells in D6-deficient politeal LNs 2 hours after subcutaneously footpad administration of LPS. (D) Intravenous administration of a CCR2 antagonist partially restored LC (CD11c⁺EpCAM⁺FITC⁺) migration from inflamed skin to draining LNs in D6-deficient mice. (E) A diagrammatic representation of the hypothesized role for D6 on LECs. (F) D6 impairs inflammatory leukocyte binding to HUVECs. Control HUVECs or HUVECs transfected with D6-GFP were activated by TNF and TAMRA-labeled THP-1 cell binding measured. Data are from 3 (C) and 2 (A-B,D,F) independent experiments with n > 3 mice per experimental group for each individual experiments.